EP0526192A2 - Hexapeptide - Google Patents

Hexapeptide Download PDF

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Publication number
EP0526192A2
EP0526192A2 EP92306942A EP92306942A EP0526192A2 EP 0526192 A2 EP0526192 A2 EP 0526192A2 EP 92306942 A EP92306942 A EP 92306942A EP 92306942 A EP92306942 A EP 92306942A EP 0526192 A2 EP0526192 A2 EP 0526192A2
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EP
European Patent Office
Prior art keywords
agent
peptide
hexapeptide
group
resin
Prior art date
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Granted
Application number
EP92306942A
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English (en)
French (fr)
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EP0526192A3 (en
EP0526192B1 (de
Inventor
Sakae c/o Tsumura & Co. Amagaya
Tadami c/o Tsumura & Co. Fujiwara
Masaki c/o Tsumura & Co. Aburada
Michio c/o Tsumura & Co. Nagasawa
Tsutomu c/o Tsumura & Co. Oyama
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Tsumura and Co
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Tsumura and Co
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Publication of EP0526192A3 publication Critical patent/EP0526192A3/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel hexapeptide and a pharmaceutically acceptable salt thereof.
  • the hexapeptide has an increasing vascular permeability suppression action, an anti-edematous action, an anti-inflammatory action, an vascular endothelial disorder amelioration action, a hypotensive action, an protease inhibition action an anti-desseminated intravasscular coagulation syndrome (DIC) action, and a wound healing action.
  • the hexapeptide can be useful as a medical product such as an anti-edematous agent, an anti-shock agent, an anti-thrombus agent, an anti-arteriosclerotic agent, an anti-allergic agent, a hypotensive agent, a wound healing agent, and an anti-inflammatory agent.
  • a therapeutical method which uses an osmotic diuretic agent to elevate a hydrostatic pressure of a blood and introduce a water in the blood.
  • a hypertonic solution of a diuretic agent such as glycerol or mannitol is quickly injected intravenously three or four times a day. Each injection time takes about 30 minutes to an hour.
  • Antibiotics are mainly used in conventional therapy for septicemia. But, since the antibiotics cannot suppress the increasing vascular increasing permeability, shocks cannot be often remedied satisfactorily in the therapy using the antibiotics.
  • Adrenal cortical hormones are also used together with the above antibiotics for suppressing the increasing vascular permeability. However, a large dose of steroids such as adrenal cortical hormones causes side effects such as an immuno-suppression, an electrolytic abnormality, and a rebound phenomenon.
  • a medicament has not yet been found which can suppress the increasing vascular permeability in the edematous and the septicemia, can cause little side effects, and not require the complicated administration for dosage.
  • an object of the present invention is to provide a novel compound which has an increasing vascular permeability suppression action, and causes little side effects, when administered.
  • Another object of the invention is to provided a pharmanceutical agent which does not require complicated administration control.
  • the present invention provides a hexapeptide represented by formula (I): A-B-Pro-C-D-E (I) where A represents an L- or D- form of arginie or lysine whose N-terminal amino group is deaminated, B represents an L- or D- form of arginine, lysine or histidine, Pro represents an L- or D- form of proline, C represents an L- or D- form of tyrosine, tryptophan, or phenylalanine, D represents an L- or D- form of valine, isoleucine, or lencine, and E represents an L- or D- form of valine, isoleucine, or lincine, one of the hydrogen atoms of the amino group of which may be substituted with a C1 to C4 alkyl group, and C-terminal carboxyl group of which may be substituted with -COOR*, -CH2OR or -CONHR wherein R* represents a C
  • the present invention also provides a hexapeptide represented by formula (II): A-B-Pro-C-D-E (II) where A represents an L- or D- form of arginie or lysine whose N-terminal amino group is deaminated, B represents an L- or D- form of arginine, lysine or histidine, Pro represents an L- or D- form of proline, C represents an L- or D- form of tyrosine, tryptophan, or phenylalanine, D represents an L- or D- from of valine, isoleucine, or lencine, and E represents a L- or D- form of valine, isoleucine, or lincine, one of the hydrogen atoms of the amino group of which may be substituted with a C1 to C4 alkyl group, and C-terminal carboxyl group of which is substituted with -COOR*, -CH2OR or -CONHR wherein R* represents
  • the hexapeptide or its salt of the invention has, in addition to a vascular permeability acceleration suppression action noted above, an anti-edematous action, an anti-inflammatory action, a vascular endothelial disorder amelioration action, a hypotensive action, a protease inhibition action, anti-DiC action, and a wound healing action.
  • the hexapeptide or its salt of the invention may be useful as an active ingredient for a pharmaceutial agent such as an anti-edematous agent, an anti-shock agent, an anti-thrombus agent, an anti-arteriousclerotic agent, an anti-allergic agent, a hypotensive agent, a wound healing agent, or an anti-inflammatory agent.
  • hexapeptide represented by formulas (I) and (II), and pharmaceutically acceptable salts thereof are sometimes referred to as hexapeptide compound of the present invention below.
  • the six amino acids which constitute the hexapeptide of the present invention may either be of L-type or D-type.
  • the first amino acid from N terminal of the hexapeptide, represented by A is an arginine or lysine whose N-terminal amino group is deaminated (Formula (I)), or alkylated or acylated (Formula (II)).
  • the alkyl group which is introduced to the N-terminal amino group of the amino acid A may be, e.g., methyl, ethyl, propyl and butyl.
  • the alkylation of the N-terminal amino group may be carried out, for example, by reacting the amino acid A with a corresponding alkyl iodide such as methyl iodide or ethyl iodide in an organic solvent such as acetone.
  • the acyl group which is introduced to the N-terminal amino group of the amino acid A may be, e.g., formyl, acethyl, propionyl, benzoyl or p-tolune sulfonyl.
  • the acylation of the N-terminal amino group may be carried out, for example, by reacting the amino acid A with an acid anhydride such as acetic anhydride or an acid chloride such as acetyl chloride in an organic solvent such as methyl chloride or pyridine.
  • the peptide bond between the fifth amino acid (represented by D) and the sixth amino acid (represented by E) from N-terminal of the hexapeptide is represented by formula (III): where X represents a hydrogen atom or C1 to C4 alkyl group.
  • the alkyl group may be metyl, ethyl, n-propyl, t-propyl, n-butyl, i-butyl or t-butyl.
  • the sixth amino acid from N terminal of the hexapeptide, represented by E is a valine, isoleusine or leusine.
  • the C-terminal carboxyl group of the sixth amino acid E may, or may not be, substituted with -COOR*, -CH2OR or -CONHR where R* represents C1 to C4 alkyl group, and R represents a hydrogen atom or a C1 to C4 alkyl group (Formula (I)).
  • the alkyl group may be, e.g., methyl, ethyl, n-propyl, t-propyl, n-butyl, i-butyl and t-butyl.
  • the C-terminal carboxyl group of the sixth amino acid E is substituted with -COOR*, -CH2OR or -CONHR mentioned above (Formula (II)).
  • substitution group to the C-terminal carboxyl group for example can be achieved by, for example, the alkylation as described above.
  • hexapeptides according to the present invention can be synthesized by a liquid- or solid-phase method which is a known method in peptide synthesis.
  • a peptide synthesis using the solid-phase method will be described in detail below.
  • the six amino acids are sequentially condensed, using an organic solvent-insoluble resin, starting from the amino acid of the C-terminal of the hexapeptide and is then treated with an acid, thereby obtaining the hexapeptide in a free form.
  • the organic solvent-insoluble resin is preferred to be chemically stable in an organic solvent and have good swell characteristic.
  • the organic solvent-insoluble resin is exemplified by a resin obtained by introducing a side functional group such as a chloromethyl group or a hydroxymethyl group into a styrene-divinylbenzene copolymer.
  • the functional group is activated prior to the synthesis of the hexapeptide.
  • the carboxy group of the amino acid E is coupled to the activated functional group of the resin.
  • a-amino group or both the ⁇ -amino group and a side-chain functional group of the amino acids which constitute the hexapeptide of the present invention is protected by a protective group, and the protected amino acids are used in the synthesis of the hexapeptide.
  • the protective group of the a-amino group are a t-butyloxycarbonyl group (Boc), a 9-fluorenylmethyloxycarbonyl group (Fmoc), or their equivalent groups.
  • Examples of a protective group for a phenolic hydroxyl group of tyrosine are a benzyl group (Bzl), a 2,6-dichlorobenzyl group (Cl2-Bzl) as a derivative thereof, an o-bromobenzyloxycarbonyl group (Br-Z), and their equivalent groups.
  • Leucine and isoleucine can be generally used as protected by the Boc or Fmoc. These protected amino acids can be commercially available products.
  • an organic solvent-insoluble resin as mentioned above must be activated. This activation can be performed as follows.
  • the resin is placed in a peptide solid-phase method reaction vessel, methylene chloride is added thereto. Then, and a 10% triethylamine (TEA)/methylene chloride mixed solution is added thereto a total of once to three times. Every time the 10% TEA/methylene chloride is added, the resultant mixture is stirred for 5 to 10 minutes. The mixture is then filtered and the resin is washed. In this activated resin, TEA (amino group) is attached to the functional group of the resin.
  • TEA amino group
  • the reaction vessel as described above may have a charge port for charging a reagent including the protected amino acids, a solvent, and the like and a filter for filtering the solvent, and may allow a reaction of the resin and the reagent by shaking of the vessel or stirring of the contents.
  • the reaction vessel is preferably made of glass or Teflon (Trade mark) and allows filtering by raising or reducing a pressure in the reaction vessel.
  • a solvent such as methylene chloride, chloroform, dimethylformamide (DMF), or benzene, which can swell the resin are added to 1 g of the resin activated as described above to obtain a suspension.
  • a C-terminal amino acid whose ⁇ -amino group is protected by the protective group with respect to one equivalent weight of the activated functional group of the resin is added in the obtained suspension.
  • the C-terminal amino acid is corresponding to the sixth amino acid from N-terminal of the hexapeptide of the present invention and is represented by E in the formulas (I) and (II).
  • the resultant mixture is stirred or shaken for about 1 to 20 minutes.
  • coupler examples include dicyclohexylcarbodiimide (DCC), water-soluble carbodiimide, carbonyldiimidazole, a Woodword's reagent "K”, N-ethyl-2-hydroxybenzisoxazolium trifluoroborate, 1-ethoxycarbonyl-2-ethoxy-1-2-dihydroxyquinoline, a "Bop reagent”, and diphenylphosphorylazido.
  • DCC dicyclohexylcarbodiimide
  • K Woodword's reagent
  • K N-ethyl-2-hydroxybenzisoxazolium trifluoroborate
  • 1-ethoxycarbonyl-2-ethoxy-1-2-dihydroxyquinoline a "Bop reagent”
  • diphenylphosphorylazido examples of the coupler are dicyclohexylcarbodiimide (DCC), water-soluble carbodiimide, carbonyldiimidazole, a Wood
  • the degree of progress of the coupling reaction can be monitored by a ninhydrin reagent or a fluorescamine test. When the reaction is not completed, coupling is repeated.
  • the amino acid whose a-amino group is protected can also be combined to a resin in accordance with an active esterification method or a symmetry anhydride method, instead of the method using the coupler as described above.
  • the resin coupled with the C-terminal amino acid (C-terminal amino acid-resin) is washed once to several times by 2 to 50 ml of a washing solvent with respect to 1 g of the resin which is activated but not couple with the amino acid, i.e., original activated resin.
  • a washing solvent for example, at least one solvent of methylene chloride, chloroform, methanol, ethanol, DMF, benzene, and acetic acid can be used.
  • the reaction product is then filtered.
  • the nonreacted, activated functional groups or amino groups of the washed resin are then blocked for prevention of further reactions, using a terminating reagent and are then washed again.
  • a terminating reagent for example, about 0.5 to 5 equivalent weight of the terminating reagent with respect to 1 equivalent weight of the functional group of the resin is added and reacted with the resin for about 10 minutes to 18 hours.
  • Examples of the terminating reagent are acetic anhydride/TEA/methylene chloride, acetic anhydride/TEA/chloroform, acetylimidazole/DMF, and fluorescamine/diisopropylethylamine/methylene chloride.
  • TFA trifluoroacetic acid
  • 100 v/v% TFA or TFA diluted to 10 v/v% or more with methylene chloride, chloroform, or their equivalents can be used, for example, as follows: About 2 to 50 ml of a TFA solution with respect to 1 g of the original activated resin are preferably added to the C-terminal amino acid-resin and reacted for about 5 to 60 minutes. After the reaction, the reaction mixture is filtered and the C-terminal amino acid-resin washed with the washing solvent.
  • piperidine is suitable as an elimination reagent for Fmoc, and used upon dilution with methylene chloride, DMF, chloroform, or their equivalent to a concentration of 5 to 50%, for example, as follows: About 2 to 100 ml of the piperidine solution with respect to 1 g of original activated resin are added to the C-terminal amino acid-resin and reacted for about 5 to 60 minutes. After the reaction, the reaction solution is filtered and the C-terminal amino acid-resin washed with the washing solvent.
  • the fifth amino acid from the N-terminal of the hexapeptide of the present invention (represented by D in the formulas (I) and (II), is coupled to the now free ⁇ -amino group of the C-terminal amino acid bonded to the resin, as follows.
  • the solvent which can swell the resin mentioned above is added to the resultant C-terminal amino acid-resin to suspend it.
  • About 1 to 6 equivalent weight of the protected fifth acid D with respect to 1-equivalent of the functional group on the resin is added to the suspension, and the coupler as described above is then added to the resultant mixture.
  • the degree of progress of the coupling reaction can be monitored by a ninhydrin reagent or a fluorescamine test. After the reaction, the reaction product is washed with the washing solvent. When the reaction is not completed, the above coupling step is repeated or blocking of excessive ⁇ -amino groups of C-terminal amino acids from subsequent reactions is performed using the terminating reagent mentioned above.
  • the the resin coupled with the peptide chain i.e, peptide-resin
  • HF hydrogen fluoride
  • a special vessel is required for the HF treatment, and is commercially available.
  • the dried peptide-resin is placed in a vessel, 0.5 to 5 ml of anisole are added thereto with respect to 1 g of peptide-resin to prevent a side reaction, and the mixture is stirred. 2 to 50 ml of a liquid HF are added to the mixture with respect to 1 g of the peptide-resin to treat the peptide-resin at -20 to 0°C for 0.5 to 2 hours.
  • Dimethylsulfide or ethanedithiol is preferably added to the anisole.
  • HF is removed at a reduced pressure, and residual HF, a protective group, anisole, and other additives are eliminated by a solvent such as ethyl acetate, diethyl ether, or benzene.
  • a solvent such as ethyl acetate, diethyl ether, or benzene.
  • the peptide is extracted using an aqueous acidic solution such as an aqueous acetic acid solution, and the resin from which the peptide is eliminated is removed by filtration.
  • the extract obtained in the above step contains, in addition to the desired peptide, byproducts such as defective peptides formed during the synthesis and must be purified.
  • the extract containing the peptide and the byproducts is concentrated by ultrafiltration. Thereafter, ion exchange, freezing, and drying are performed thereby obtaining a dried product.
  • the dried product is purified by a fractional reverse-phase high-performance liquid chromatography. Finally, ion exchange and gel filtration of a fraction containing the desired peptide are performed to obtain a purified hexapeptide according to the present invention.
  • Examples of a pharmaceutically acceptably, non-toxic salt of the hexapeptide according to the present invention are salts with an alkali metal such as sodium or potassium or with an alkaline earth metal such as calcium and magnesium, and acid addition salts with an inorganic acid such hydrochloric acid, sulfuric acid, phosphoric acid or carbonic acid, or with an organic acid such as acetic acid propionic acid, tartaric acid succicinic acid, malic acid, asparatic acid or glutamic acid.
  • the phermaceutically acceptably salt of the hexapeptide according to the present invention also includes a complex salt with a metal compound such as a zinc, a nickel or a cobalt compound, and with a polyamic acid such as poly-L-gultamic acid.
  • a metal compound such as a zinc, a nickel or a cobalt compound
  • a polyamic acid such as poly-L-gultamic acid
  • the hexapeptide compound according to the present invention directly act on vascular endothelial cells, it is effective and can strongly suppress an edema associated with the increasing vascular permeability.
  • the hexapeptide compound can suppress edemata based on vascular endothelial disorders and various tissue disorders in addition to suppression of a cerebral edema.
  • the hexapeptide compound according to the present invention has also an anti-endotoxin shock effect, protease inhibition action (an anti-thrombin action, an anti-plasmin action), a hyoptensive action, an anti-DIC action, an anti-allergic action, and a wound healing action.
  • the hexapeptide compound according to the present invention can storlongly suppress the increasing vascular permeability and do not exhibit any side effect around in steroids. Therefore, the hexapeptide compound is useful for diseases such as a cerebral edema, an edema of the lung, an edema of the trachea, a thrombus, an arteriosclerosis, a burn, and a hypertension, and allergic diseases such as a bronchial astham and a pollenosis.
  • diseases such as a cerebral edema, an edema of the lung, an edema of the trachea, a thrombus, an arteriosclerosis, a burn, and a hypertension
  • allergic diseases such as a bronchial astham and a pollenosis.
  • the hexapeptide compound according to the present invention is useful as agents for reducing hemorrhage from a sharp trauma such as an injured tissue portion at the time of surgical operation, a lacerated wound of a brain or other tissues caused by a traffic accident and the like, and for relaxing and curing swelling, pain and inflammation caused by the traumata.
  • the hexapeptide compound according to the present invention can also be useful for suppressing internal hemorrhage caused be a dull trauma, and edemata and inflammation which are accompanied with the internal hemorrhage.
  • the hexapeptide compound according to the present invention also provides excellent effects in suppression and improvement of cerebral edemata by suppressing a leakage of blood components to a tissue matrix found in cerebral ischemetic diseases which include cerebral infractions (e.g., a cerebral thrombus and a cerebral embolism), intracranial hemorrhages (e.g., a cerebral hemorrhage and a subarachnoidal hemorrhage), a transient cerebral ischemic attack, and a acute cerebral blood vessel disorders in a hypertensive encephalopahty.
  • cerebral infractions e.g., a cerebral thrombus and a cerebral embolism
  • intracranial hemorrhages e.g., a cerebral hemorrhage and a subarachnoidal hemorrhage
  • transient cerebral ischemic attack e.g., a transient cerebral ischemic attack
  • the hexapeptide compound according to the present invention provides effects in suppression and improvement of burns, chilblains, other skin inflammations and swelling, an upper tracheal inflammation, an asthma, nasal congestion, a pulmonary edema, and inflammable disorders caused by endogenous and exogenous factors, which directly damage vascular endothelia and mucous membranes, such as an environmental chemical substance, chemotherapeutics of cancer, an endotoxin, and an inflammation mediator.
  • the hexapeptide compound according to the present invention can reduce the hemorrhage, inflammation, swelling and pains of a sharp trauma, i.e., the tissue portion injured by a surgical operation and a traffic accident.
  • the hexapeptide compound also suppresses internal hemorrhage caused by a dull trauma and edemate and inflammation which are accompanied with the internal hemorrhage.
  • the hexapeptide compound according to the present invention is effective in treatments of cerebral ischemetic diseases which contain cerebral infarctions (e.g., a cerebral thrombus and a cerebral embolism), intracranial hemorrhages (e.g., a cerebral hemorrhage and a subarachnoidal hemorrhage), a transient cerebral ischemic attack, and acute cerebral blood vessel disorders in a hypertensive encephalopathy.
  • cerebral infarctions e.g., a cerebral thrombus and a cerebral embolism
  • intracranial hemorrhages e.g., a cerebral hemorrhage and a subarachnoidal hemorrhage
  • a transient cerebral ischemic attack e.g., a transient cerebral ischemic attack
  • acute cerebral blood vessel disorders in a hypertensive encephalopathy edemata are produced during in these acute cerebral blood vessel disorders, suppression of edemata greatly influences the recovery after curing the
  • the pharmaceutical agent according to the invention containing a hexapeptide compound as an active ingredient, can be used as a medicine used in therapy of the above-mentioned diseases.
  • the pharmacetical agents of the invention is especially good for an anti-edematous agent, an anti-shock agent, an anti-thrombus agent, an anti-arteriosclerosis agent, an anti-allergic agent, a hypotensive agent, a wound healing agent, and an anti-inflammatory agent.
  • the hexapeptide compound according to the invention and the pharmaceutical agents can be administered either orally or parenterally in an amount effective for therapy.
  • the effective amount of the hexapeptide compound of the invention, per day is between 0.1 and 150 nmol/kg.
  • the administration amount of the hexapeptide compound should be determined depending on a variety of factors such as degree of disorders, weight of the patient, and age.
  • the pharmacetical agent of the invention may contain pharmaceutically acceptably diluent or excipient, in the form of liquid, gel, or solid, other than the hexapeptide compounds. Further, if needed, the agent may contain an additive which can be generally used in a drug composition, such as a general antiseptic agent, anti-oxidation agent, or the like.
  • the pharmaceutical agent of the invention can be used as an oral or parenterally administration drug.
  • the oral administration drug takes the form of, for example, regular tablet, capsule, powder, solution, or suspension
  • the parenteral administration drug is in the form of, for example, regular solution injection, suspension injection, suppository, or nasal mucosal spray.
  • the agent is preferably administered through intravenous or hypodermic injection.
  • the form of the agent can be selected depending on condition of the patient, age, and degree of disorders.
  • hexapeptide numbers are corresponding hexapeptides shown in Table 1 and 2.
  • desamino-Arg represents ⁇ -guanidinopentanic acid.
  • N-acetyl- and N-buthyl- represent acetylated and butylated derivatives of a N-terminal amino acid of the hexapeptide, respectively.
  • -OH represents that a carboxylic group of a C-terminal amino acid is free
  • -OEt represents that the carboxylic group is converted into an ethylester
  • -NH2 represents that the carboxylic group is converted into a carbamoyl.
  • Leucinol is an alcohol form of leucine, i.e., -CH2OH group replaces the carboxylic group of leucine.
  • washing operation 1.5 l of methylene chloride, 1.5 l of methanol, and 1.5 l of methylene chloride were added to the activated resin in the order named, and stirred for 2 minutes. The above operation was repeated twice, and the resultant solution was filtered off (this washing operation will be referred to as a washing operation I hereinafter).
  • Boc- represents that the a-amino group of an amino acid is protected by a protective group Boc.
  • HOBt represents 1-hydroxybenzotriazole.
  • Arg used in step 7 was performed as following. First, 0.1 mol of 5-aminovaleric acid hydrochloride was dissolved in 50 ml of a 2N-sodium hydroxide solution. 0.1 mol of S-methylthiocarbamide was added thereto, and the resultant solution was stirred and reacted at room temperature for 2 days. The reaction mixture was concentrated and purified by a column chromatography. The purified product was converted to hydrochloride. The Desamino-Arg hydrochloride obtained as described above used in step 7.
  • Step 8 Elimination of Peptide from Resin and Elimination of Protective Group
  • Ether was added to the solution, and the resultant solution was stirred well and filtered using a glass filter.
  • the product remaining on the filter was washed with ether (a total of about 300 ml), and a peptide eliminated from the resin was extracted four times using 100 ml of a 0.1N aqueous acetic acid solution.
  • the pH of the extract obtained in step 8 was adjusted to 5.0 by acetic anhydride, and filtered using an ultrafiltration membrane having a molecular weight of 1,000.
  • the resultant solution was subjected to a column of a cation exchange resin (available from Whatman). Using an ammonium acetate buffer as a mobile phase, the component absorbed on the resin was eluted. The eluted component was freeze-dried.
  • the dried product was purified by a fractional reverse-phase high-performance liquid chromatography.
  • this chromatography use was made of an ODP-90 (available from ASAHI CHEMICAL INDUSTRY CO., LTD.) as the column and a phosphate buffer/acetonitrile mixed solvent as gradient eluent.
  • a main peak fraction obtained by the above chromatography was concentrated by the ultrafiltration membrane as mentioned above.
  • the obtained concentrate was subjected to column of cation exchange resin SP-Sephadex (available from Pharmacia Fine Chemicals, Inc.).
  • SP-Sephadex available from Pharmacia Fine Chemicals, Inc.
  • aqueous sodium chloride solution as a mobile phase
  • the component adsorbed on the resin was eluted.
  • the eluted component was subjected to again a column of Sephadex G-25 (available from Pharmacia Fine Chemicals, Inc.).
  • an aqueous acetic acid solution as a mobile phase
  • the resultant product exhibited a single spot by a thin layer chromatography using three developing solvents and electromigration (Whatman 3MM, pH 3.5; 1,500 v. 1hr.).
  • Hexapeptides of samples having peptide Nos. 2 to 10 were synthesized and purified following the same procedures as in Example 1 except that the starting amino acids were changed to the protected amino acids corresponding to the peptide compositions shown in Table 1.
  • NCH3 represent a peptide bond whose hydrogen atom was substituted by methyl group (The same applies below).
  • each peptide-resin (500 mg) obtained by sequentially condensing source amino acids by the same procedures as Example 1 was suspended in 1N TEA/ethanol (50 ml), and the resultant mixture was stirred at room temperature for 17 hours. The resultant mixture was filtered and the resin was washed with ethanol. The filtrate and the washing solution were collected and concentrated at a reduced pressure. The resultant crude products were purified as in of Example 1.
  • Lys 1.02 (1); Pro: 0.98 (1); Trp: 1.03 (1); Ile: 0.97 (1); Leu: 0.97 (1); NCH3: present (1); Desamino-Arg NH3: - (1)
  • each peptide chain (0.02 mol) was dissolved in a solvent mixture of 30 ml of water and 30 ml of dioxane, and sodium bicarbonate (0.048 mol) and Boc-N3 (0.024 mol) were added thereto. The resultant solution was stirred and reacted at 40 to 45°C for 24 hours.
  • the reaction mixture was concentrated at a reduced pressure, 50 ml of water were added thereto, and the resultant mixture was washed with 50 ml of ethyl acetate. The water layer was collected, and 70 ml of 0.5M citric acid were added thereto with icing. Sodium chloride was added to the resultant solution to be saturated. The reaction product was extracted with 100 ml ethyl acetate three times and was then dried with sodium sulfate. The dried product was concentrated at a reduced pressure.
  • the peptide chains (0.005 mol) was dissolved in 10 ml of THF, and 0.45 ml of N-methylformate and 0.35 ml of ethyl chlorocarbonate were added thereto while the solution was cooled to -20°C. After a lapse of 5 minutes, 5 ml of a DMF solution containing leucinol hydrochloride (0.005 mol) and N-methylformate (0.45 ml) were added to the above solution, and the resultant mixture was stirred and reacted at -70°C for 2 hours. As result, leucinol was condensed to the peptide chain.
  • the synthesized peptide chain was eliminated from the resin, and the protective group was eliminated from the peptide chain as in Example 1.
  • the resin was washed with a washing solvent.
  • the washing solution and the filtrate were concentrated and the obtained concentrate was freeze-dried.
  • the a-amino group of the peptide chain contented in dried product was protected with a Boc.
  • Leucinol was then condensed to the peptide chain, and the protective group was eliminated from the hexapeptide thus obtained.
  • the crude product was purified with HPLC.
  • the result supported the resultant product obtained by the above method was peptide No. 32 having the amino acid composition shown in Table 2.
  • the result supported the resultant product obtained by the above method was peptide No. 33 having the amino acid composition in Table 2.
  • vascular endotherial disorder amelioration action an anti-edematous action, an anti-inflammatory action, an anti-shock action, an anti-DIC action, a protease inhibition action, anti-allergic action a hypotensive action, and a wound healing action of the hexapeptide compound according to the present invention.
  • SD rats 6-week male Sprague-Dawley (SD) rats (each group consisted of 10 rats) were fed for a week, they were used in this experiment.
  • the concentrations of samples of the hexapeptides (peptide Nos. 1 to 30) were dissolved by an acetic acid buffer solution added with a 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Rate of Increase in Leg volume (%) (Heated Leg Volume) - (Non-heated Leg Volume) (Heated Leg Volume) x 100
  • the peptides Nos. 1 to 30 were confirmed to have a vascular permeability acceleration suppression action and could suppress edemata.
  • An action of the hexapeptide compounds according to the present invention for suppressing the increasing vascular permeability is a lowering of a permeability of vascular endothelial cells to close a gap between the vascular endothelial cells.
  • arteriosclerotic diseases as shown in a hypercholesterolemia model of a monkey proposed by R. Ross (pp. 103 - 108, 1990, Elsevier Science Publisher B.V., Netherlands), monocytes, T-lymphocytes, and denatured cholesterols are attached to the gaps between the vascular endothelial cells and intrude under the vascular endothelial cells through the gaps, thereby finally forming fatty spots as an initial change in morbid state in an arteriosclerosis.
  • vascular endothelial cells are thinned with an increase in fatty spots and are finally separated and open. Thereafter, most of foamy cells flow out, and platelets are attached to cause a thrombus. Smooth muscle cells are grown by a PDGF (Platelet-Derived Growth Factor) produced from the platelets, and the wall thickness of the artery is increased to result in an arteriosclerosis.
  • PDGF Platinum-Derived Growth Factor
  • LDL low-density lipoprotein
  • mice each group consisted of 10 mice each having a weight of 25 g to 35 g were used.
  • Each of peptides Nos. 1, 31 and 32 was dissolved in a sodium acetate buffered solution (pH 6.0) added with 1% BSA or in a physiological saline.
  • An endotoxin to induce a shock was obtained by dissolving Lipopolysaccharide [E.coli; 0127B8 (Difco)] in a physiological saline.
  • peptialdes Nos. 1, 31 and 32 were intravenously injected at a by dose of 5 ml/kg to each mouse, and the endotoxin with concentration of 10 mg/kg was intravenously injected at a dose of 5 ml/kg after 10 minutes. The lethal rates (%) of these mice within 24 hours were measured.
  • 5 ml/kg of the sodium acetate buffered solution added with 1% BSA was injected instead of the hexapeptide of the present invention.
  • a septic shock is a major cause of death of the mouse.
  • the septic shock is an acute circulatory disorder which exhibits a shock state such that a vascular endothelial disorder occurs by bacterial infection by means of an endotoxin serving as a bacterial cell wall component, and increasing vascular permeability is to cause leakage of blood components outside the blood vessel.
  • Iron column whose diameter was 5 mm cooled by dryice acetone was placed on the left epidural portion of each rat to cause a vascular cerebral edema. Freezing was performed to form an injured portion after an hour, and the brain of each rat was taken out. The brain was divided into right and left cerebral cortices, and weights of the left cerebral cortices were measured. The left cerebral cortices was dried at 105°C for 24 hours, and weights of the dried left cerebral cortices were measured to obtain water contents (%).
  • the peptides Nos. 1, 31 and 32 were intravenously injected at the concentration shown in Table 6 to each rat 10 minutes before and 20 minutes after the freezing.
  • 1 ml/kg of the saline added with 1% BSA instead of the peptides was injected into rat according to same procedures as described above.
  • the peptides Nos. 1, 31 and 32 according to the present invention were confirmed to have the increasing vascular permeability suppression action and suppress the edemata.
  • Hybrid grown-up dogs (each group consisted of six dogs) were used in this experiment.
  • Peptide No. 1 was dissolved in a sodium acetate buffered solution added with a 1% BSA. 25 mg/kg of sodium pentobarbital were intravenously injected to each dog.
  • a blood pressure of the femoral artery and a blood flow rate of the ascending aorta of each dog were measured while performing the artificial respiration. 2 nmol/kg of peptide No. 1 was intravenously injected. Thereafter, the blood pressure and the blood flow were measured again.
  • the blood flow rate was decreased from 0.95 l/min to 0.75 l/min by the injection.
  • the hexapeptide compounds according to the present invention have a hypotensive action and can serve as useful as hypotensive agent by reducing the resistance in peripheral blood vessels.
  • Hybrid grown-up dogs (each group consisted of six dogs) were used in this experiment. 30 mg/kg of pentobarbital were intravenously injected to anesthetize each dog. Intravenous injection of 32 nmol/kg of each of peptides Nos. 1, 31 and 32 was performed. After 30 minutes, 3 mg/kg of an endotoxin derived from Escherichia Coli were intravenously injected while the artificial respiration was performed with air.
  • Samples of blood were collecting from each dog immediately before the injection of the peptides and 120 minutes after the injection of the endotoxin.
  • the number of platelets is the number per 1 mm3 (1 ⁇ l) of the blood in Table 7.
  • the hexapeptides according to the present invention were confirmed to suppress a blood coagulation acceleration state and a decrease in the number of platelets.
  • peptides Nos. 1, 31 to 34 were mixed at various volume with thrombin (final conc. 10 U/ml), and the resultant mixture was preincubated at 37°C for 5 minutes.
  • a synthetic coloring base (Test Team R S-2238 available from Daiichi Kagaku Yakuhin) was added to the above preincubated mixture and was reacted at 37°C for 15 minutes.
  • 2% citric acid was added to the above mixture to stop the reaction. Hydrolysis was performed using residual thrombin, and free p-nitroaniline removed from the synthetic coloring base was subjected to colorimetric determination at a wavelength of 405 nm, thereby obtaining the inhibition capability with reference to 0.1M tris buffer. On the base of the inhibition capability thus obtained, a concentration of the peptides when each peptide inhibited the activity of thrombin with 50%, i.e. IC50 was calculated.
  • the hexapeptide compounds according to the present invention were confirmed to have an anti-thrombin action as an inhibition action against a protease.
  • An anti-egg albumin rat serum was diluted with a saline to forty-fold of its volume, thereby obtaining an anti-egg albumin rat serum solution.
  • 1 ml of the anti-egg albumin rat serum solution was intradermally injected to both side parts against the median line in the shaved portion, thereby sensitizing passively the rats.
  • 1 ml of a 0.5% Evans blue solution containing 10 mg of a egg-albumin was intravenously injected to the each rat, thereby provocating a passive cutaneous anaphylaxis (PCA) reaction.
  • PCA passive cutaneous anaphylaxis
  • the rats were decapitated, thereby subjecting to hemorrhage to death.
  • the skins were peeled from the rats, and a long diameter and a short diameter of a blue stained portions caused by the PCA reaction in each rat were measured, and area of the blue-stained portions were calculated on the base of the obtained data.
  • the hexapeptide compounds according to the present invention were confirmed to have an anti-allergic action, and were useful for an anti-allergic agent.
  • hexapeptide compounds according to the present invention were useful for an anti-inflammatory agent, since the inflammatory originates in the disorders of the vascular endothelim and the tissular mucous membranes caused by e.g., various parameters, which were eliminated by the increasing vascular permeability suppression.
  • the male SD rats (each group consisted of 10 rats) were anesthetized with ether and shaved in the back of the each rat, and the anesthetized portion was formed by a length of 30 mm along the median line by using a degreased, sterilized razor blade.
  • the wound was immediately sutured at three equidistant positions, and the stitches were taken out after three days.
  • the rats were subjected to hemorrhage to death in the eighth day, and the skins were peeled from the rats.
  • a skin stripe having a size of 1 cm x 4 cm was formed from a wounded portion of each rat. Both ends of this stripe were set in a wound curing measurement tensile test instrument (TK-251 available from Unicom), and a tensile force (traction tension: g/cm) required to cut the wound portion was measured.
  • TK-251 wound curing measurement tensile test instrument
  • Each hexapeptide compound according to the present invention was dissolved in an acetic acid buffer solution added with 1% BSA and was intravenously injected to each rat for 8 days (once a day) from the date of wound formation.
  • acetic acid buffer solution added with 1% BSA
  • 1 ml/kg of the sodium acetat buffered solution added with 1% BSA instead of the hexapeptide solutions was injected according to same procedure as described above.
  • the hexapeptide compounds were confirmed to promote wound healing of the skins of the rats.
  • Peptide No. 1 obtained in Example 1 injection distilled water, sodium chloride, and gelatin were used to obtain an injection in accordance with a conventional injection manufacturing method.
  • Peptide No. 5 obtained in Example 5 sodium chloride, sodium acetate, hydrochloric acid, methyl para-hydroxybenzoate, ethyl para-hydroxybenzoate, propyl para-hydroxybenzoate, and butyl para-hydroxybenzoate were used to obtain an injection in accordance with the conventional injection manufacturing method.
  • Example 6 An aqueous solution containing peptide No. 6 obtained in Example 6 and mannitol was freeze-dried. An aqueous solution containing gelatin and phenol was added to the dried product to obtain an injection.
  • Peptide No. 8 obtained in Example 8 and cacao butter or Whitepsole were used to obtain a suppository in accordance with a conventional suppository manufacturing method.
  • Peptide No. 11 obtained in Example 11 and gavakisate methanesulfonate were used to form a capsule in accordance with a conventional capsule manufacturing method.

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EP92306942A 1991-07-30 1992-07-30 Hexapeptide Expired - Lifetime EP0526192B1 (de)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007705A1 (en) * 1993-09-15 1995-03-23 The Regents Of The University Of California Method for inhibiting vascular leakage and anti-inflammatory compounds
WO1996019494A1 (en) * 1994-12-19 1996-06-27 Biochem Pharma Inc. Peptides having immunomodulatory activity
US5846940A (en) * 1994-08-05 1998-12-08 Sinseiro Okamoto Corneal therapeutic agent
EP2895186A4 (de) * 2012-09-04 2016-05-18 Univ Leland Stanford Junior Therapeutische zusammensetzungen und zugehörige verfahren
WO2023186193A1 (en) * 2022-03-31 2023-10-05 Contipro A.S. Hexapeptide, composition comprising thereof and topical use thereof

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US5955430A (en) * 1993-09-24 1999-09-21 University Of Southern California Use of angiotensin II fragments and analogs thereof in tissue repair
US6214790B1 (en) 1998-04-10 2001-04-10 Mayo Foundation For Medical Education And Research Neo-tryptophan
US7087575B2 (en) * 2002-07-18 2006-08-08 Mayo Foundation For Medical Education And Research Treating the effect of nicotine
US20080139481A1 (en) * 2004-06-17 2008-06-12 Dix Thomas A Non-Natural Amino Acids
WO2006085417A1 (ja) * 2005-02-10 2006-08-17 Nagase Chemtex Corporation 生活習慣病を予防する食品
WO2007100718A2 (en) * 2006-02-24 2007-09-07 Denise Barbut Neurotensin receptor agonists and opioid receptor agonists
WO2010085661A1 (en) * 2009-01-23 2010-07-29 Musc Foundation For Reasearch Development Modified peptides and their use
CN105541642A (zh) * 2016-01-11 2016-05-04 罗梅 一种手性（s）-亮氨醇盐酸盐的合成方法

Citations (1)

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EP0333071A2 (de) * 1988-03-11 1989-09-20 Eisai Co., Ltd. Polypeptide, deren Methoden zur Herstellung, sie enthaltende pharmazeutische Zusammensetzungen und Verwendung

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US4411890A (en) * 1981-04-14 1983-10-25 Beckman Instruments, Inc. Synthetic peptides having pituitary growth hormone releasing activity
US4110321A (en) 1976-07-12 1978-08-29 Folkers Karl Synthetic tridecapeptide [Gln4 ]-neurotensin having hormonal activity
US4425269A (en) * 1982-06-07 1984-01-10 Merck & Co., Inc. Metabolically protected analogs of neurotensin
IT1190389B (it) * 1985-09-19 1988-02-16 Eniricerche Spa Esapeptidi ad attivita' ipotensiva
JP2714425B2 (ja) * 1988-03-11 1998-02-16 エーザイ株式会社 ポリペプチド

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0333071A2 (de) * 1988-03-11 1989-09-20 Eisai Co., Ltd. Polypeptide, deren Methoden zur Herstellung, sie enthaltende pharmazeutische Zusammensetzungen und Verwendung

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Title
EUR. J. BIOCHEM. vol. 124, 1982, pages 117 - 125 GRANIER, C. ET AL. 'Synthesis and Characterization of Neurotensin Analogues for Structure/Activity Relationship Studies' *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007705A1 (en) * 1993-09-15 1995-03-23 The Regents Of The University Of California Method for inhibiting vascular leakage and anti-inflammatory compounds
US5846940A (en) * 1994-08-05 1998-12-08 Sinseiro Okamoto Corneal therapeutic agent
WO1996019494A1 (en) * 1994-12-19 1996-06-27 Biochem Pharma Inc. Peptides having immunomodulatory activity
US5980913A (en) * 1994-12-19 1999-11-09 Biochem Pharma, Inc. Peptides having immunomodulatory activity
EP2895186A4 (de) * 2012-09-04 2016-05-18 Univ Leland Stanford Junior Therapeutische zusammensetzungen und zugehörige verfahren
WO2023186193A1 (en) * 2022-03-31 2023-10-05 Contipro A.S. Hexapeptide, composition comprising thereof and topical use thereof

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KR930002373A (ko) 1993-02-23
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JP2803477B2 (ja) 1998-09-24
DK0526192T3 (da) 1997-07-28
AU2065792A (en) 1993-02-04
KR100251496B1 (ko) 2000-05-01
AU661003B2 (en) 1995-07-13
US5393740A (en) 1995-02-28
DE69217763T2 (de) 1997-07-10
EP0526192B1 (de) 1997-03-05
ES2101037T3 (es) 1997-07-01
ATE149518T1 (de) 1997-03-15
DE69217763D1 (de) 1997-04-10
CA2074967A1 (en) 1993-01-31
JPH05194590A (ja) 1993-08-03

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